Serum microRNAs targeting ACE2 and RAB14 genes distinguish asymptomatic from critical COVID-19 patients

Despite the extraordinary advances achieved to beat COVID-19 disease, many questions remain unsolved, including the mechanisms of action of SARS-CoV-2 and which factors determine why individuals respond so differently to the viral infection. Herein, we performed an in silico analysis to identify host microRNA targeting ACE2, TMPRSS2, and/or RAB14, all genes known to participate in viral entry and replication. Next, the levels of six microRNA candidates previously linked to viral and respiratory-related pathologies were measured in the serum of COVID-19-negative controls (n = 16), IgG-positive COVID-19 asymptomatic individuals (n = 16), and critical COVID-19 patients (n = 17). Four of the peripheral microRNAs analyzed (hsa-miR-32-5p, hsa-miR-98-3p, hsa-miR-423-3p, and hsa-miR-1246) were upregulated in COVID-19 critical patients compared with COVID-19-negative controls. Moreover, hsa-miR-32-5p and hsa-miR-1246 levels were also altered in critical versus asymptomatic individuals. Furthermore, these microRNA target genes were related to viral infection, inflammatory response, and coagulation-related processes. In conclusion, SARS-CoV-2 promotes the alteration of microRNAs targeting the expression of key proteins for viral entry and replication, and these changes are associated with disease severity. The microRNAs identified could be taken as potential biomarkers of COVID-19 progression as well as candidates for future therapeutic approaches against this disease.


INTRODUCTION
On 30 January, 2020, the World Health Organization declared coronavirus disease 19 (COVID-19) a public health emergency of international concern, becoming a global pandemic on 11 March, 2020. 1 COVID-19 has drastically affected the entire world, at both economic and public health levels, with millions of deaths worldwide. 2,3 The enormous effort in the development of vaccines against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has significantly decreased the rhythm of disease progression. Unfortunately, it is still not possible to predict the efficacy and durability of immunity after vaccination, mainly taking into account the viral mutations constantly arising, as in the case of Omicron, one of the last variants identified. 4,5 Thus, major efforts are actually focused on understanding the molecular mechanisms of SARS-CoV-2 infection, as well as on the identification of therapeutic targets against COVID-19 and potential biomarkers to prevent its progression. 6 Two clear candidates in this therapeutic approach are angiotensin-converting enzyme 2 (ACE2), one of the main co-factors required by SARS-CoV-2 to access human host cells, and the transmembrane protease serine 2 (TMPRSS2), which is responsible for priming of the viral spike protein, a step required to allow the virus-host cell membrane fusion and further internalization of the virus. 7,8 ACE2 and TMPRSS2 have received more attention; however, other host-cell proteins related to SARS-CoV-2 replication are also playing an essential role during viral infection, promoting its survival. For instance, the interaction of SARS-CoV-2 with host proteins involved in the assembly and viral trafficking, such as Ras-related protein Rab-14 (RAB14), may favor SARS-CoV-2 replication. 9,10 Hence, the use of agents blocking or interfering with the interaction between SARS-CoV-2 and ACE2, TMPRSS2, or RAB14 might help to prevent or reduce the virus entry and replication. Indeed, some inhibitors are being tested. That is the case of camostat mesylate, targeting TMPRSS2, which seems to block SARS-CoV-2 infection of lung cells. 8 Alternatively, the use of inhibitors against SARS-CoV-2 mRNA translation are also of interest. 11,12 In this regard, the application of specific mi-croRNAs (miRNAs) to regulate the expression of essential proteins for the SARS-CoV-2 infection process constitutes a promising but also unexplored approach. 13,14 MiRNAs are small (18-22 nucleotides) highly conserved, non-coding single-stranded ribonucleic acids (RNAs), which appear to be involved in many physiological processes as well as in different diseases, participating, among others, in the modulation of viral infection and host defense. 15 To date, several miRNAs have been found in altered levels in the blood of individuals after viral infection, including COVID-19 patients. 6,16,17 Thus, miRNAs not only constitute promising therapeutic tools 17 but they are also considered as potential prognostic markers for SARS-CoV-2 infection. 18 In this study, based on the literature and in silico results, we focus on analyzing the levels of miRNA targeting ACE2, TMPRSS2, and RAB14, all associated with viral entry and replication in the serum of COVID-19 patients. Our results indicate that peripheral hsa-miR-32-5p, hsa-miR-98-3p, hsa-miR-423-3p, and has-miR-1246 increased in critical COVID-19 patients. The functional pathways in which these molecules participate, as well as the potential use of these molecules as biomarkers, are discussed.
Next, the number of binding sites of all six miRNAs were analyzed, indicating the number of all selected miRNAs in the 3 0 UTR of ACE2, TMPRSS2, and RAB14 (Table S2). As previously described, 19 a match of six nucleotides between miRNA and 3 0 UTR sequence may be considered as a potential seeding region ( Figure S1). hsa-miR-98-3p and hsa-miR-423-3p displayed the highest number, with six 3 0 UTR binding sites for the RAB14 gene. Furthermore, the target genes of these six miRNA candidates were also predicted ( Figure 1C). According to protein-protein interaction networks analysis of the shared target genes ( Figure 1D), they all displayed a narrow interaction with each other (p = 0.003).
Diagnostic potential of serum miRNAs and the association with critical COVID-19 patients Next, to analyze the diagnostic value, the area under the curvereceiver operating characteristic (AUC-ROC) was compared for the single and combined differentially expressed miRNAs ( Table 2). Although the combined miRNA AUC values were significant, none of them reached a value higher than single miRNAs. The ROC curve of single hsa-miR-32-5p, hsa-miR-98-3p, hsa-miR-423-3p, and hsa-miR-1246 revealed the probability to use them as valuable biomarkers to diagnose critical COVID-19 patients from COVID-19-negative controls and asymptomatic IgG-positive individuals ( Figure 4A-D). The highest discriminatory power achieved by a single miRNA was acquired for hsa-miR-1246, with an AUC of 0.875 (95% CI: 0.755-0.995; p = 0.0002) ( Figure 4D). Finally, the association between peripheral miRNA levels and clinical variables was also evaluated ( Table 3). The circulating hsa-miR-32-5p and hsa-miR1246 levels showed a negative correlation with the presence of risk factor. Furthermore, the circulating hsa-miR-32-5p levels indicated a positive correlation with the age and severity of COVID-19, which could be possible since miRNA expression is related to age. 20 On the other hand, although a differential expression of miR-NAs has been described between males and females, only the levels of hsa-98-3p showed an association with the gender. 21 Functional enrichment analysis A total of 2,812 genes were detected as potential targets of our four differentially quantified serum miRNAs. Remarkably, RAB14 was the only common target gene for hsa-miR-32-5p, hsa-miR-98-3p, hsa-miR-423-3p, and hsa-miR-1246 ( Figure 5A). According to the functional analysis performed via the KEGG database ( Figure 5B), the genes targeted by these four miRNAs were mainly involved in the processing of genetic information, cell signaling, and other cellrelated processes (extended in Figure S3).
Moreover, the targeted genes were involved in several biological processes ( Figures 6A-6E), which were all significantly enriched (extended in Table S4). Within the cellular process category, many genes correlated with vesicle trafficking and cell-cell interactions, as well as regulation of cell cycle, cell proliferation, migration, or even regulation of the NLRP3 inflammasome complex ( Figure 6A). Many genes were involved in RNA transcription and mRNA processing and transport, which correlates with the involvement of miRNAs in the regulation of RNA polymerase I and II expression, mRNA processing, stability, and splicing, among others ( Figure 6B). Moreover, these genes were also directly linked to the viral process, including viral RNA replication and viral entry into the host nucleus or regulation of the viral genome replication ( Figure 6D). Also, all four miR-NAs have been previously associated to respiratory-related diseases (extended in Table S5). Finally, several genes were associated to blood-related processes, such as coagulation, vessel development, morphogenesis, and remodeling ( Figure 6C), and also to neuroepithelial cell differentiation and regulation of neural precursor cell proliferation, among others ( Figures 6D and 6E).

DISCUSSION
After 2 years since the first person was diagnosed with COVID-19, more than 447 million cases have been reported around the world, including more than 6 million deaths. 22 To date, numerous diagnostic methods and vaccines have been developed in record time, thanks to extraordinary and unprecedented scientific and clinical efforts. Nevertheless, the mechanisms of action of SARS-CoV-2, as well as the potential secondary effects that this virus exerts over the organism are not fully understood yet.
One of the unanswered questions regarding COVID-19 is why some people infected with SARS-CoV-2 present severe symptoms, even though others do not. Most patients show mild to moderate symptoms such as fever, persistent dry cough, body aches, and occasional dyspnea. However, a small fraction of patients may also present acute respiratory failure and acute respiratory distress syndrome associated with multiple organ failure. 23,24 Consequently, knowing an individual's susceptibility to SARS-CoV-2 infection, through identifying critical biomarkers, may guide future treatment strategies for the disease and its different phases. In this sense, peripheral miRNAs have been remarked as potential biomarkers for COVID-19 treatment and diagnosis since they can play an essential role in the pathogenesis of its infection. 6,25 In this study, we focused on the identification of miRNAs targeting ACE2, TMPRSS2, and RAB14 genes, due to their direct or indirect association with SARS-CoV-2 infection. While other miRNAs had been recently associated with the pathology of COVID-19, being upregulated in acute and critical patients compared with controls (i.e., miR-29a-3p, -146a-3p, -155-5p, 25 or miR-6501-5p and miR-618 6 ), none of them targeted ACE2, TMPRSS2, or RAB14. Notably, four of the six miRNAs selected from the in silico analysis (hsa-miR-32-5p, hsa-miR-98-3p, hsa-miR-423-3p, and hsa-miR-1246) were significantly increased in the serum of critical COVID-19 patients. In addition, hsa-miR1246 could discriminate between IgG-positive asymptomatic subjects and critical COVID-19 patients. Furthermore, based on their AUC-ROC values, these four miRNAs could be considered novel biomarkers with high-yield diagnostic accuracy. In addition, hsa-miR-32-5p and hsa-miR-1246 correlated with the presence of obesity, 26,27 AHT, 28 T2D, 29 asthma, 30 or thalassemia, 31 which have been described as risk factors for critical COVID-19 patients.
Remarkably, none of the four miRNAs found altered in the serum of critical COVID-19 patients had TMPRSS2 as a target gene, while they all targeted RAB14. Among them, hsa-miR-32-5p has been associated in silico to RAB14 in pancreatic and colon tumors. 32 Moreover, hsa-miR-32-5p and miR-98-3p, also targeting RAB14, were identified as crucial lung cancer-associated miRNAs, [33][34][35] and their deregulation levels were identified in several respiratory disorders, such as acute respiratory distress syndrome, 36,37 bronchopulmonary dysplasia, 38 and congenital pulmonary airway malformations. 39 Besides, these miR-NAs have been previously related with viral processes. For instance, miR-32-5p has a significant regulatory role in avian hepatitis A viral infection, 40 visna maedi virus, 41 and caprine arthritis encephalitis viruses, 41 while the deregulation of the expression miR-98 has been described in a mouse model of West Nile virus neuropathogenesis. 42 Apart from these two miRNAs, hsa-miR-423-3p also exhibited RAB14 as a unique target gene. While previous studies found increased levels of the circulating 5 0 form (hsa-miR-423-5p) in COVID-19 patients, nothing was described concerning hsa-miR-423-3p. 43 This miRNA has been defined as a biomarker of lung cancer, 44 and it has also been related to tuberculosis and endocytosis pathways, in which the RAB protein family plays a crucial role. 45 Finally, according to our in silico analysis, hsa-miR-1246 was the only miRNA that targeted both ACE2 and RAB14 genes. As mentioned above, hsa-miR-1246 not only discriminated critical COVID-19 patients versus COVID-19-negative controls, it also allowed to distinguish critical versus asymptomatic patients, in agreement with a recent study in which has-miR-1246 appeared differentially expressed in severe versus asymptomatic COVID-19 patients, although this study was performed only with male patients. 46 Notablhashsa-miR-1246 has been described as a biomarker of emphysema in patients with chronic obstructive pulmonary disease, 47 and also with nonsmall cell lung cancer progression. 48 Moreover, miR-1246 has been identified as a possible regulator of the SARS-CoV-2 genome, which would provide more information on the protection mechanisms associated with miRNAs. 49 Concerning ACE2 expression, a preliminary study described that the ACE2 mRNA levels were inversely proportional to miR-1246 levels in the airways epithelium of smokers. 50 Noteworthy, in our study population most of our critical COVID-19 patients were non-smokers. Moreover, Khan and co-workers described, both in silico and in vitro, that ACE2 was regulated by miR-1246 in patients with acute respiratory distress syndrome. 51,52 Finally, as regards Rhas4, hsa-miR-1246 has been also described as a potential prognostic biomarker for glioma, being predicted as one of its target genes. 53 In addition to ACE2 and RAB14, another 2,810 target genes were predicted for our 4 miRNAs. Overall, our in silico analysis indicated that these miRNAs are much more than simple post-transcriptional regulators since, besides, they have been involved in several biological processes related to the pathogenesis of SARS-CoV-2. For instance, the increase seen of these four circulating miRNAs may correlate, for example, with the decreased levels of RAB14 transcripts found in lung biopsies from patients with adenocarcinomas, 54 together with many other crucial genes for SARS-CoV-2 infection. RAB14 participates in the formation of vesicles 55 necessary for the maturation and assembly of the structural proteins of SARS, 9,10 therefore it might also constitute an essential protein for infection by SARS-CoV-2. 56 In this sense, the analysis of the biological and functional roles indicated that not only RAB14 but many other targeted genes appear involved in vesicular trafficking, cell-cell interactions, and regulation of the cell cycle, among others. In addition, these genes also participate in viral entry into the host cell, viral RNA replication, or even regulation of viral genome replication, which, together with the possible inhibition of the expression of RAB14 by the increase of the four miRNAs, could indicate a defense mechanism of the cells against the proliferation of the virus. Finally, several genes were also associated to regulation of the NLRP3 inflammasome complex, which has been linked to the severity of COVID-19, 57 and also with blood-and coagulation-related processes. This might correlate with the hypercoagulability and thrombotic events that take place in response of COVID-19. 58

Conclusions
In this study, we have identified four miRNAs targeting ACE2 and/or RAB14 that could be taken as potential biomarkers of COVID-19 progression, allowing to distinguish critical patients from asymptomatic and negative individuals. The identified miRNA have been previously associated to respiratory-related diseases, including SARS-CoV. Moreover, many other gene targets of these miRNA have been associated with viral replication and inflammation-and coagulationrelated processes. The individuals included in this study were recruited before being vaccinated, so any potential effect that vaccines might have over our results should be further evaluated. On the other hand, the limited access to serum samples in our study population constitutes a clear limitation in our study. Ideally, a higher number of samples should be analyzed to further validate the specificity of these miRNAs as biomarkers. Besides, even though peripheral miRNA levels were quantified, there was no confirmation about the direct secretion from the respiratory tissues into the extracellular space in COVID-19 patients, although previous research has shown their presence in the lungs. Future studies should include in vitro and in vivo models of COVID-19 to corroborate the bioinformatic predictions. These analyses might confirm the involvement of this four-miRNA panel as prognostic markers of SARS-CoV-2 infection, as well as their potential role as therapeutic candidates to inhibit the host response against this or other related viruses.

MATERIALS AND METHODS
Bioinformatic analysis to predict miRNAs that target ACE2, TMPRSS2, and RAB14 The miRDB database (http://mirdb.org/) and TargetScan (http:// www.targetscan.org) were used to predict the miRNAs and their targeted genes, 19,59 and the network image was obtained using the miR-Net (https://www.mirnet.ca) tool. 60 All these analyses were performed using the default parameters, and Homo sapiens was selected as the specific Taxonomy. The shared target genes of the predicted miRNAs were analyzed with STRING, an on-line platform to identify functional protein-association networks (https://string-db.org), 61 while the miRNA-gene network image was obtained with Flourish software (https://flourish.studio). Finally, STarMiR (www.sfold.wadsworth. org/cgi-bin/starmir.pl) was used for the prediction of miRNA binding sites to 3 0 UTR mRNA binding sites (seeding region).

Study population
In total, 49 subjects were included in this study. Based on qPCR analysis against SARS-CoV-2, and ELISA tests for specific IgG and IgM antibodies (IME00136 and IME00137, Erba Mannheim), subjects were classified into three groups: (1) COVID-19-negative controls, which were PCR and IgG negative at the time of serum extraction (n = 16), (2) asymptomatic COVID-19 individuals, PCR negative and IgG positive at the time of serum extraction (n = 16), and (3) critical COVID-19 patients (n = 17) who required hospitalization. The first two groups (COVID-19-negative controls and asymptomatic donors) were enrolled at the National Paraplegic Hospital (Toledo, Spain), between April and May 2020. Critical COVID-19 patients were recruited at the time of hospitalization at the University Hospital Puerta del Mar (Cadiz, Spain) in July 2021, and the COVID-19 Hospital (Seville, Spain) in May 2021. The protocol was approved by the ethics committee at each center, and the study was conducted in accordance with the Helsinki II Declaration. Only donors older than 18 years were included in the study, and written informed consent was provided by all of those who participated in this study.

Serum collection
Peripheral blood samples were collected with serum separator tubes (SST II Advance, BD Vacutainer). The blood was then mixed up and down 8-10 times. After that, SST tubes were incubated at room temperature for at least 30 min to ensure the separation of the serum from the cellular components, and the serum was collected by centrifugation (2,000 Â g for 10 min at 4 C). Serum samples were aliquoted and stored at À80 C until further use.

RNA isolation
Isolation of total RNA, including miRNAs, was performed with 200 mL of serum using the miRNeasy Serum/Plasma Kit (QIAGEN) following the manufacturer's instructions. Before purification, each serum sample was spiked (RNA spike-in, QIAGEN) with UniSp2 (2 fmol), UniSp4 (0.02 fmol), UniSp5 (0.00002 fmol), and MS2 RNA (Merck) to monitor the technical quality of RNA isolation according to the manufacturer's guidelines. Directly after isolation, RNA was subjected to the reverse transcription process.

Reverse transcriptase reaction
An MiRCURY LNA Reverse Transcription (RT) Kit (QIAGEN) was used to synthesize cDNA according to manufacturer's instructions. Isolated RNA (2 mL) were added to the reaction tube to make up a final volume of 10 mL reaction mix. UniSp6 (0.075 fmol) and cel-miR-39-3p (0.001 fmol) were used as positive controls for cDNA synthesis (QIAGEN). The reaction took place for 60 min at 42 C, heat inactivated for 5 min at 95 C, and immediately cooled to 4 C in a thermal cycler. Then cDNA samples were stored at À20 C.

Real-time quantitative polymerase chain reaction analysis of miRNAs expression levels
Samples from the RT reaction were prepared with the miR-CURY SYBR Green PCR Kit (QIAGEN) and assessed for miRNA gene expression using the miRCURY LNA miRNA Serum/Plasma Focus PCR Panels (QIAGEN) according to the manufacturer's protocol. The interpolate calibrator UniSp3 was used to account for the variability between plates. Real-time qPCR analysis was performed in the CFX Connect PCR System (Bio-Rad) at 95 C for 2 min to heat samples, followed by 40 cycles of 95 C for 10 s, and 56 C for 60 s, followed by melting curve analysis. The analyzed miRNAs primer information can be found in Table S6. qPCR amplification curves were evaluated with CFX Manager software (Bio-Rad). The specificity of the amplification was confirmed by the melting curve analysis. Then, the expression level of each miRNA was calculated using the 2 ÀDCq method (where DCq = CqmiRNA À Cq hsa-miR-103a-3p). The normalized miRNA levels were further log2 converted.

Functional enrichment analysis
Functional enrichment of predicted genes of differentially expressed miRNAs was analyzed using FunRich software (http://www.funrich. org/). The KEGG, GO, Uniprot, Reactome, and FunRich databases were used to identify the molecular functions and biological processes. Finally, the miRNA TissueAtlas (https://ccb-web.cs.unisaarland.de/tissueatlas) was used to determine the expression of the selected miRNA within the tissues.

Statistical analysis
All statistical analyses were performed using GraphPad Prism 8 software. Comparisons among multiple groups were performed using one-way analysis of variance, followed by non-parametric Kruskal-Wallis rank tests. ROC curves were applied to characterize the diagnostic performance of both each and combined miRNAs. ROC curves were generated by plotting sensitivity against 100% specificity, indicating the AUC and 95% confidence intervals. Pearson correlation coefficient was used for correlations between log2 miRNAs versus clinical parameters in critical COVID-19 patients. Differences were considered statistically significant at p < 0.05.

DECLARATION OF INTEREST
The authors declare no competing interests.